Molecular oxygen is a critical requirement for cellular function in animals, while the protection from O.sub.2 is required for the function of plant enzymes such as the nitrogenases. Animals, and mammals in particular, are critically dependent upon the continuous supply and utilization of O.sub.2 in the processes which maintain life. This is particularly true for high O.sub.2 -consuming tissues such as neural tissues and muscle, both striated and smooth. Interruption of oxygen delivery to these tissues, for times as brief as minutes, can result in cell death and loss of functions critical to organ function. Moreover, it is the oxygen concentration within tissues, rather than within blood or other bodily fluids, which ultimately supports cellular function. Therefore, the measurement of bodily fluid and tissue oxygen concentrations is of pivotal importance to clinical medicine, with the compromise of oxygen delivery to tissues occurring in a host of vascular diseases, such as arteriosclerosis, diabetes, sickle cell disease, and impaired wound healing to name a few. It is therefore not surprising that numerous patents have been issued for methods and devices which measure blood and bodily fluid oxygen concentration, and this inventor's U.S. Pat. No. 5,186,173 (hereinafter the Zuckerman '173 patent) for the first measurement of tissue oxygen concentration in vivo.
These patents, although each addressing problems involved in the noninvasive determination of bodily fluid or tissue PO.sub.2, suffer from deficiencies which have precluded their widespread application in clinical medicine. To achieve the requirement of noninvasive measurement recent patents have turned to optical methods, which involve the quenching of phosphorescence or fluorescence of a dye by dioxygen. For example, patents such as that of U.S. Pat. No. 4,476,870 (Peterson et. al.) have developed catheter designs in which a fluorescent substance, such as perylene, is housed within a sealed catheter which may be inserted into blood vessels and its fluorescence quenching by O.sub.2 in blood measured by use of fiber optics. This device, although providing the ability to measure bodily fluid or blood PO.sub.2, suffers from two deficiencies. First, the PO.sub.2 of blood may be measured accurately only after the catheter probe is externally calibrated prior to its insertion into a blood vessel; and second, the technique provides no topographic information, as PO.sub.2 is measured at a single locus, viz., at the probe tip. In U.S. Pat. No. 4,810,655, Khalil et. al. purport to overcome the need for prior calibration of the catheter before its every usage by measuring phosphorescence lifetime instead of intensity. Although in theory a direct lifetime system should overcome the need for prior calibration, in practice, as shown in Table 4 of the Khalil et. al. patent, the phosphorescence lifetimes of the porphyrins employed change with light exposure, making calibration prior to use still necessary. In addition, the time-resolved direct lifetime system would be cumbersome to implement, and less precise than a steady-state approach. Vanderkooi and Wilson (U.S. Pat. No. 4,947,850) developed a procedure based upon the determination of the phosphorescence lifetime of an O.sub.2 -sensitive probe substance, such as a metallo-porphyrin bound tightly to albumin, which phosphoresces on a timescale of fractions of a millisecond (10.sup.-3 sec), and whose phosphorescence lifetime is reduced (quenched) by dioxygen. Here the O.sub.2 -sensitive probe is a phosphorescing molecule which may be injected into the blood stream, thereby permitting topographic determination of the PO.sub.2 of blood within the vasculature of an imaged tissue. However, due to self-quenching of the metallo-porphyrins, as well as the other probes described by Vanderkooi, these probe molecules cannot be used alone. That is, self-quenching results in probe concentration dependent changes in decay time which are greater than those induced by molecular oxygen. Since it is impossible to know the precise probe concentration in the blood, due to leakage at the injection site and variations in blood volume in different animals or humans, they must modify the probe to eliminate self-quenching. As stated in U.S. Pat. No. 4,947,850 (col. 3, line 15) "porphyrins are preferably employed and said compositions are preferably admixed with proteinaceous compositions which bind with the phosphorescent composition . . . Albuminous . . . compositions are preferred." As described in their publication Vanderkooi, J. et. al., "An Optical Method for Measurement of Dioxygen Concentration Based Upon Quenching of Phosphorescence", J. Biol. Chem., 262(12): 5476-5482 (April 1987), the technique will simply not work without albumin or some other large molecular mass protein bound to the probe molecule. When injected intravenously into the blood the large molecular mass protein (M.W.=67,000) prevents the probe molecule from passing through the small junctions or lipid membranes of the blood vessel wall, and thereby limits the measurement to oxygen concentration in the blood. Similarly, U.S. Pat. No. 4,579,430 (Bille) discloses an invention that allows the determination of oxygen saturation, percentage binding of O.sub.2 to blood hemoglobin, within retinal vessels. In a host of diseases the oxygen saturation and oxygen concentration of the blood, as revealed by previous patents, remains normal although the tissue is believed to become hypoxic. Such diseases include diabetes, retinopathy of prematurity, and hypertensive and arteriosclerotic diseases. Similarly, during long surgeries, such as bypass surgery, carotid artery surgery and during prolonged intensive care, the oxygen saturation and/or concentration of the blood is carefully monitored and maintained, yet moderate to severe brain damage due to hypoxia has been known to occur. It is the oxygen concentration within tissues which is relevant to its functioning and health, and tissue oxygen concentration depends upon tissue oxygen consumption rate, blood velocity, and vessel caliber, in addition to blood oxygen concentration or saturation. Therefore, it is of paramount importance to be able to measure the PO.sub.2 of tissue in space and time.
The noninvasive, topographic measurement of tissue PO.sub.2 was addressed in the the Zuckerman '173 patent. In this patent, a highly lipid soluble probe substance, such as sodium pyrenebutyrate, is injected intravenously or intraperitoneally, or applied topically when appropriate. The lipid soluble, biocompatible probe substance leaves the blood and accumulates within the lipid bilayers of tissue cells. Here the probe is essentially the tissue itself, once pyrenebutyrate accumulates within the lipid bilayers of its cells. This and the digital imaging system detailed within the patent allows the first topographic determination of tissue oxygen concentration, which supports tissue health and function. The concentration of O.sub.2 is determined in the Zuckerman '173 patent by the measurement of fluorescence intensity, according to the Stern-Volmer equation, written in terms of fluorescence intensities. The invention of the Zuckerman '173 patent, although allowing the first noninvasive determination of tissue PO.sub.2 in space and time, suffers from two deficiencies which preclude its clinical application, and which presently limits its utility to research applications on laboratory animals. The most significant limitation results from the fact that fluorescence intensity is determined by both the concentration of pyrenebutyrate in space within tissues, which varies due to spatial differences in lipid composition of the cells within tissues, as well as by the spatial distribution of tissue oxygen concentration. To circumvent this problem, and to extract the oxygen concentrations at a plurality of locations, the fluorescence intensity at each locus is ratioed against the fluorescence intensity of the same location when the tissue is brought to a PO.sub.2 of 0 mm Hg (in the absence of oxygen). This may be accomplished by breathing the animal on 100% N.sub.2 at the end of the experiment. Such a procedure cannot be applied clinically on humans as it would undoubtedly result in cell death, evidenced in brain damage or death. The second deficiency in the invention of the Zuckerman '173 patent resides in the optical filtering effects of blood on the fluorescence intensities at the emission wavelength of pyrenebutyrate. This may similarly be corrected in a laboratory situation by the separate measurement of the optical density of blood within the vasculature of the imaged tissue.
Both of the deficiencies in the device and method of the Zuckerman '173 patent may be obviated by the direct measurement of fluorescence lifetime (decay) instead of fluorescence intensity. Measurement of fluorescence lifetime at a given PO.sub.2 and within a given tissue, once determined in a laboratory calibration of the instrument, would obviate the need to bring the tissue to a PO.sub.2 of 0 mm Hg in the clinic, as the fluorescence decay constant is independent of pyrenebutyrate concentration, and is similarly unaffected by the absorbance of blood within the vasculature. However, fluorescence lifetime occurs on the timescale of nanoseconds (10.sup.-9 see), and in addition to the cumbersome and expensive apparatus required directly to measure such short fluorescence lifetimes at a plurality of locations, would require intense pulsed laser excitation (pulse duration &lt;20 nanoseconds) of the tissue at the ultraviolet excitation wavelength to achieve usable signal-to-noise ratios. Since the quantum efficiency of pyrenebutyrate and other fluorescent probe molecules is generally less than 0.5, photon absorption which is not converted into emitted radiation by fluorescence would be converted to heat, thereby causing tissue damage. Tissue damage subsequent to an intense laser pulse, used in a time-resolved direct fluorescence lifetime determination, is the major drawback to the use of this approach in vivo. Herein resides the need for a method for the topographic measurement of oxygen concentration in vivo by a steady-state, rather than time-resolved, procedure which provides an indirect determination of fluorescence lifetime.
In view of the deficiencies of the prior art, it would be desirable to have a method which can be applied in numerous forms and which allows the determination of bodily fluid, blood, and tissue PO.sub.2 in space and time by an indirect determination of fluorescence lifetime. Such a method would allow the construction of an O.sub.2 -sensitive catheter, which may be inserted into a blood vessel, and which does not require cumbersome external calibration prior to its every usage. Similarly, such a method would, by intravenous injection of a biocompatible lipid soluble fluorescent dye, and by the application of digital image processing techniques, allow the topographic determination of tissue PO.sub.2, as well as the determination of blood PO.sub.2 within the vasculature of the imaged tissue. By extension, the application of optical serial sectioning methodologies would allow the first determinations of tissue and blood PO.sub.2 distributions in three dimensions, tomographically within a volume of imaged tissue. Most importantly, such a procedure would be eminently suitable for clinical applications involving diagnosis and treatment of vascular, metabolic and other diseases in humans.